The term "Campylobacter" as used herein, refers to the bacteria classified as such in Bergey's Manual of Systematic Bacteriology, Vol. 1 (N. R. Krieg and J. G. Holt [eds. ], 1986, pp.111-118, , Williams & WilKins). Detection of Campylobacter is important in various medical and public health contexts. Campylobacter jejuni and C. coli are the two most important species, causing diarrhea (Blaser et al., 1979, Ann. Intern, Med. 91:179), and enteritis (G. K. Morris et al., eds, American Society for Microbiology, Washington, D.C.) in humans. Other Campylobacter species have been implicated in causing disease in humans or animals, such as abortion, septicemia and proliferative ileitis. In addition, microorganisms resembling Campylobacter have been isolated from feces of homosexual men (Fennell et al., 1984 J. Infec. Dis. 149:58) and from gastric ulcer biopsies (Kasper et al., 1984 Infection. 12:179).
It is, therefore, an aspect of the present invention to provide a novel assay system capable of rapidly detecting Campylobacter and which is generally applicable to environmental, food or clinical samples.
Campylobacter generally are identified pursuant to a standard laboratory method (Campylobacter, In Washington, J. A. [ed.], Laboratory Procedures in Clinical Microbiology, 2nd Ed., New York, Springer-Verlag, 1985, pp. 215-217).
It is yet another aspect of the present invention to avoid the disadvantages associated with traditional culturing techniques and to employ nucleic acid probes to detect Campylobacter.
It is yet another aspect of the present invention to provide probes which can hybridize to target regions which can be rendered accessible to the probes under normal assay conditions.
While Kohne et al. (1968, Biophysical Journal 8:1104-1118) discuss one method for preparing probes to rRNA sequences they do not provide the teaching necessary to make Campylobacter-specific probes.
Pace and Campbell (1971, Journal of Bacteriology 107:543-547) discuss the homology of ribosomal ribonucleic acids from diverse bacterial species and a hybridization method for quantitating such homology levels. Similarly, Sogin, Sogin, and Woese (1972, Journal of Molecular Evolution 1:173-184) discuss the theoretical and practical aspects of using primary structural characterization of different ribosomal RNA molecules for evaluating phylogenetic relationships.
Fox, Pechman, and Woese (1977, International Journal of Systematic Bacteriology 27:44-57) discuss the comparative cataloging of 16S ribosomal RNAs as an approach to prokaryotic systems. These references, however, fail to relieve the deficiency of Kohne's teaching with respect to Campylobacter.
Rashtchian and Fitts in copending application U.S. Ser. No. 692,778 now U.S. Pat. No. 4,785,086, discuss the generation of certain Campylobacter-specific nucleic acid probes targeted at genomic DNA sequences, but the method described would not yield small oligonucleotide probes.
It is yet another aspect to provide small oligonucleotide probes capable of specifically detecting Campylobacter. Romaniuk et al. (1987, J. Bacteriol. 169:2137-2141), Rashtchian et al. (1987, Current Microbiol. 14:311-317), Lau et al. (1987, System and Appl. Microbiol. 9:231-238), Paster and Dewhirst (1988, Intnl. J. System. Bacteriol. 38:56-62), and Thompson et al. (1988, Intnl. J. System. Bacteriol. 38:190-200) discuss Campylobacter ribosomal RNA gene organization and present 16S rRNA sequences from various Campylobacter. The references, however, fail to identify to probe target regions of most interest.
Romaniuk and Trust (1987, FEMS Microbiol. Lett. 43:331-335), describe an oligonucleotide probe which hybridizes to a region of Campylobacter 16S rRNA and demonstrates its use in identifying strains of Campylobacter by Southern hybridization to electrophoretically-separated restriction fragments of Campylobacter genomic DNA. While useful in this limited context, this probe does not have sufficient specificity to identify Campylobacter in samples containing mixed populations of Campylobacter and non-Campylobacter bacteria.
Ribosomes are of profound importance to all organisms because they serve as the only Known means of translating genetic information into cellular proteins, the main structural and catalytic elements of life. A clear manifestation of this importance is the observation that all cells have ribosomes.
Ribosomes contain three distinct RNA molecules which, at least in E. coli, are referred to as 5S, 16S, and 23S rRNAs. These names historically are related to the size of the RNA molecules, as determined by sedimentation rate. In actuality, however, they vary substantially in size between organisms. Nonetheless, 5S, 16S, and 23S rRNA are commonly used as generic names for the homologous RNA molecules in any bacteria, and this convention will be continued herein.
Hybridization is traditionally understood as the process by which, under predetermined reaction conditions, two partially or completely complementary single-stranded nucleic acids are allowed to come together in an antiparallel fashion to form a double-stranded nucleic acid with specific and stable hydrogen bonds. The stringency of a particular set of hybridization conditions is defined by the base composition of the probe/target duplex, as well as by the level and geometry of mispairing between the two nucleic acids. Stringency may also be governed by such reaction parameters as the concentration and type of ionic species present in the hybridization solution, the types and concentrations of denaturing agents present, and/or the temperature of hybridization. Generally, as hybridization conditions become more stringent, longer probes are preferred if stable hybrids are to be formed. As a corollary, the stringency of the conditions under which a hybridization is to take place (e.g., based on the type of assay to be performed) will largely dictate the preferred probes to be employed. Such relationships are well understood and can be readily manipulated by those skilled in the art. As a general matter, dependent upon probe length, such persons understand stringent conditions to mean approximately 35.degree. C.-65.degree. C. in a salt solution of approximately 0.9 molar.
As used herein, probe(s) refer to synthetic or biologically produced nucleic acids (DNA or RNA) which, by design or selection, contain specific nucleotide sequences that allow them to hybridize under defined predetermined stringencies, specifically (i.e., preferentially) to target nucleic acid sequences.
A target nucleic acid sequence is one to which a particular probe is capable of preferentially hybridizing.
Still other useful definitions are given as their first use arises in the following text. All references cited herein are fully incorporated by reference.